Angiogenesis, the process of developing a hemovascular network, is essential for the growth of solid tumors and is a component of normal wound healing and growth processes. It has also been implicated in the pathophysiology of atherogenesis, arthritis, and diabetic retinopathy. It is characterized by the directed growth of new capillaries toward a specific stimulus. This growth, mediated by the migration of endothelial cells, may proceed independently of endothelial cell mitosis.
The molecular messengers responsible for the process of angiogenesis have long been sought. Greenblatt and Shubik (J. Natl. Cancer Inst. 41: 111-124, 1968) concluded that tumor-induced neovascularization is mediated by a diffusible substance. Subsequently, a variety of soluble mediators have been implicated in the induction of neovascularization. These include prostaglandins (Auerbach, in Lymphokines, Pick and Landy, eds., 69-88, Academic Press, New York, 1981), human urokinase (Berman et al., Invest Opthalm. Vis. Sci. 22: 191-199, 1982), copper (Raju et al., J. Natl. Cancer Inst. 69: 1183-1188, 1982), and various "angiogenesis factors".
Angiogenesis factors have been derived from tumor cells, wound fluid (Banda et al., Proc. Natl. Acad. Sci USA 79: 7773-7777, 1982; Banda et al., U.S. Pat. No. 4,503,038), and retinal cells (D'Amore, Proc. Nat. Acad. Sci. USA 78: 3068-3072, 1981). Tumor-derived angiogenesis factors have in general been poorly characterized. Folkman et al. (J. Exp. Med. 133: 275-288, 1971) isolated a tumor angiogenesis factor from the Walker 256 rat ascites tumor. The factor was mitogenic for capillary endothelial cells and was inactivated by RNase. Tuan et al. (Biochemistry 12: 3159-1365, 1973) found mitogenic and angiogenic activity in the nonhistone proteins of the Walker 256 tumor. The active fraction was a mixture of proteins and carbohydrate. A variety of animal and human tumors have been shown to produce angiogenesis factor(s) (Phillips and Kumar, Int. J. Cancer 23: 82-88, 1979) but the chemical nature of the factor(s) was not determined. A low molecular weight non-protein component from Walker 256 tumors has also been shown to be angiogenic and mitogenic (Weiss et al., Br. J. Cancer 40: 493-496, 1979). An angiogenesis factor with a molecular weight of 400-800 daltons was purified to homogeneity by Fenselau et al. (J. Biol. Chem. 256: 9605-9611, 1981), but it was not further characterized. Human lung tumor cells have been shown to secrete an angiogenesis factor comprising a high molecular weight carrier and a low molecular weight, possibly non-protein, active component (Kumar et al., Int. J. Cancer 32: 461-464, 1983). Vallee et al. (Experientia. 41: 1-15, 1985) found angiogenic activity associated with three fractions from Walker 256 tumors. Tolbert et al. (U.S. Pat. No. 4,229,531) disclose the production of angiogenesis factor from the human adenocarcinoma cell line HT-29, but the material was only partially purified and was not chemically characterized. Isolation of genes responsible for the production of angiogenesis factors has not heretofore been reported at least in part due to the lack of purity and characterization of the factors.
Isolation of angiogenesis factors has employed high performance liquid chromatography (Banda et al., ibid); solvent extraction (Folkman et al., ibid); chromatography on silica gel (Fenselau et al., ibid), DEAE cellulose (Weiss et al., ibid), or Sephadex (Tuan et al., ibid); and affinity chromatography (Weiss et al., ibid).
Recently, Vallee et al. (U.S. patent application Ser. No. 724,088, filed Apr. 17, 1985, and U.S. Ser. No. 778,387, now U.S. Pat. No. 4,727,137, filed concurrently with this application, both of which are hereby incorporated by reference) have purified an angiogenic protein from a human adenocarcinoma cell line. The purified protein, known as angiogenin, was chemically characterized and its amino acid sequence determined.
Because angiogenesis factors play an important role in wound healing (Rettura et al., FASEB Abstract #4309, 61st Annual Meeting, Chicago, 1977) and may find applicability in the development of screening tests for malignancies (Klagsburn et al., Cancer Res. 36: 110-114, 1976; and Brem et al., Science 195: 880-881, 1977), it would clearly be advantageous to produce angiogenic proteins in sufficient quantities to permit their application in therapy and diagnosis. The techniques of genetic engineering are ideally suited to increase production levels of these proteins. The cloning of genes encoding angiogenic proteins is a necessary first step in such large-scale production.
Furthermore, it may in some instances be desirable to obtain these proteins from non-tumor cells, such as in the case of human therapeutics, where contamination with certain tumor products would be unacceptable. This invention therefore provides for the production of angiogenic proteins in non-tumor cells using recombinant DNA techniques.